Hepatitis C virus antigen polypeptide, production method therefor, and antibody detection method

ABSTRACT

A hepatitis C virus antigen polypeptide having a molecular weight of approximately 22 kilodaltons expressed from a hepatitis C virus structural gene region; a production method for a hepatitis C virus antigen polypeptide having a molecular weight of 22 kilodaltons and/or a peptide related thereto, wherein an expression vector having inserted thereinto a cDNA fragment of a hepatitis C virus structural gene region is inserted into a cultured cell line and the transfected cell line thus obtained is cultured; and a detection method for a hepatitis C virus antibody, wherein a hepatitis C virus antigen polypeptide is used as an antigen, and an antibody specific thereto is detected.

DETAILED DESCRIPTION OF THE INVENTION

1. Technical Field

The present invention relates to a novel antigen which is useful in thediagnosis of the hepatitis C and the like and is translated from thestructural region of the hepatitis C virus genome, a production methodtherefor, and a detection method for antibodies associated withhepatitis C using this antigen.

In greater detail, the present invention relates to a novel HCV antigenpolypeptide (hereinafter termed "p22") which is a peptide which is codedfor in the hepatitis C virus (hereinbelow termed "HCV") structural generegion and which exhibits a molecular weight in SDS-PAGE of roughly 22kilodaltons, a production method for this HCV antigen polypeptide, and adetection method for antibodies associated with hepatitis C using thispolypeptide.

2. Prior Art

HCV has recently been identified as a pathogenic virus causing hepatitisC, which is one type of vital hepatitis. It is characteristic in that itis responsible for almost all cases of hepatitis C occurring after bloodtransfusions in Japan, and in that it causes not only transientinfection but also persistent infection (in Japan, about 1.2% of thepopulation is persistently infected). Approximately half of all cases ofacute hepatitis C become chronic, and furthermore, chronic hepatitis maygradually develop into cirrhosis or liver cancer. In addition, thepercentage of those who are persistently affected is reported to be from1-3% of the population worldwide. That is to say, hepatitis C is a graveinfectious disease worldwide, and the prevention, early diagnosis, andtreatment thereof have global significance.

HCV is a positive strand RNA virus;the virus genome thereof has a sizeof approximately 10,000 bases. A structural gene region coding for theviral structural protein is located on the 5' side of the genome, and anon-structural (NS) gene region is located downstream from this. Noantigen protein coded for by this virus has been identified as yet. Thesole measuring method for antigens and antibodies which has beenreported to date is an antibody (anti-C100 antibody) detection methodfor a fusion protein (C100) which is produced in yeast and contains 363amino acids of a part of the HCV non-structural gene region (a regionfrom NS3 to NS4). Using this antibody detection method, it is possibleto determine, to a certain extent, whether a history of exposure to HCVexists, so that this detection method has been used in the diagnosis ofthe hepatitis C virus. Furthermore, blood which registers positive onthis detection test often contains infectious HCV, so that thisdetection method is presently in use in Japan in the screening of bloodto be used in transfusions.

Problems to Be Solved by the Invention

The C100 antibody described above normally takes from 3-6 months tobecome positive after the infection of hepatitis C virus, so that thismethod cannot be used as a diagnostic method for hepatitis C during thisperiod, and this has been recognized to be a major problem. Furthermore,even in cases in which only blood which was negative for the anti-C100antibody was used in blood transfusions, a certain number of occurrencesof the hepatitis C virus was noted, so that it is thought that onlyapproximately half of hepatitis cases occurring after blood transfusionscould be screened out by using this method alone; there is thus a needfor a new antibody test or a detection method for virus structuralprotein antigens. Furthermore, as the C100 antigen is derived fromnon-structural protein genes, identification of a virus structuralprotein and establishment of a detection system for the antigens and theantibodies thereof is extremely important in order to find a more directdiagnostic method or candidates for future vaccines.

Means Used to Solve the Problems and Effects of the Invention

As a result of conducting research in order to solve the above problems,the present inventors have expressed the cDNA of the HCV structural generegion in cultured cells, and, by means of the fluorescent antibodytechnique and the Western blot method using the serum of hepatitis Cpatients, have discovered a novel polypeptide (p22) which is derivedfrom an HCV gene, has a molecular weight of approximately 22kilodaltons, and which reacts specifically with the serum of hepatitis Cpatients, and by separating this, have arrived at the present invention.

Next, a method was developed for the extraction and purification of theHCV-related antigen p22 from a novel cell line obtained by means oftransfecting a cultured cell line with an expression vector havinginserted thereinto the cDNA fragment of the HCV structural gene regionused above and cloning the cultured cell line, and furthermore, a methodwas developed for the extraction, purification, and efficient productionof HCV p22 in large amounts in cultured cell systems by means oftransfecting cells of a cultured cell line Sf from a cutworm moth with aBaculovirus expression vector having inserted thereinto the cDNAfragment of this HCV structural gene region.

Furthermore, using the HCV p22 obtained in this manner, an ELISA kit forthe detection of the p22 antibody was constructed, and it was determinedthat this was greatly superior to the conventional HCV antibodymeasurement method.

Furthermore, the present invention provides not merely the p22 having amolecular weight of approximately 22 kilodaltons described above, butalso an HCV-related polypeptide having a larger molecular weight andcharacterized by containing this complete peptide as a part thereof, aproduction method therefor, and an HCV antibody detection method usingthis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plasmid structural diagram showing the regions on the HCVgenome of the HCV-cDNA fragment integrated into the expression plasmidspSR316, pSR312, and pSR316d46.

FIG. 2 is a schematic diagram showing the results of the Western blot ofthe endoproteins of a cell transformed with the various plasmids ofExample (2). The "+" symbol indicates a sample which was treated withtunicamycin (Tm), while the "-" symbol indicates a sample which was nottreated with Tm.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, detailed explanation will be given concerning the p22 andp22-related polypeptides, the production method thereof, and thedetection of HCV antibodies using these antigens of the presentinvention.

(1) p22

From experimental results such as the genetic configuration of therelated Flaviviruses, the fact that the proteins thereof are rich inbasic amino acids, and the fact that they have no N-sugar chain, it isbelieved that the p22 of the present invention is a nucleocapsid proteinwhich is one of the hepatitis C virus structural proteins. Judging fromthe example of the related Flaviviruses, it is believed that thestructural protein of HCV is produced by being cut from a precursorprotein with an enzyme (signalase) which is present in animal and insectcells.

As a result of the research of the present inventors, it was discoveredthat the p22 of the present invention is a polypeptide produced by meansof the processing in the cell of a precursor polypeptide of HCV which iscoded for by the HCV structural gene. As a result of this, it isbelieved that the p22 of the present invention is a polypeptide which isidentical to the original nucleocapsid protein of HCV, and it is thoughtto be an extremely important antigen for the detection of HCV-relatedantibodies. Furthermore, the antigen of the present invention is animportant material not merely for the detection of this type ofantibody, but also for the production of a vaccine for the purpose ofpreventing HCV infection.

From the expression experiments of the present inventors using variousHCV cDNA fragments, it is conjectured that the p22 of the presentinvention is a polypeptide which is positioned at the N-terminus (aminoterminus) of the extremely long HCV precursor polypeptide for which theHCV gene codes. Furthermore, on the basis of the base sequence, it ispresumed that this polypeptide is hydrophilic and is rich in basic aminoacids.

Furthermore, the amino acid region which is from approximate the 180thto the 190th amino acid, counting from the initial amino acid(methionine) of the above precursor polypeptide has an amino acidsequence which is particularly strongly hydrophobic, so that it isbelieved that the amino acids of this region are recognized bysignalase, and the polypeptide of the present invention is produced bybeing cut from the precursor polypeptide.

(2) Production Method of p22 and Related Peptides

The p22 of the present invention can be obtained in large amounts bymeans of the expression in cultured cells of the cDNA of the the HCVstructural genetic region. It is possible to use, as the cultured cellswhich function as the host cells, animal cells such as COS cells or CHOcells, which are in common use, and the present invention may be carriedout according to standard transformational methods and culturingmethods.

On the other hand, in the case in which E. coli or a yeast, which do notpossess signalase identical to that of animal cells, are used as theexpression hosts, only the genetic region coding for p22 is put into anopen reading frame form in advance, and expression can be conducted sothat there is no need for processing after expression. However, in thecase in which expression is conducted using cDNA which is longer thanthe genetic region which codes for the p22 polypeptide, it is believedthat processing by means of signalase cannot be achieved, so that it isnot believed that p22 can be easily expressed by means of expression inE. coli or a yeast.

In order to yet further efficiently express p22, it is possible to use aBaculovirus as a vector, and to introduce this into insect cells.

Any virus which is classified as a Baculovirus may be used as this typeof virus vector; for example, Autographa Californica, Trichoplusia ni,Rachiplusia ou, Galleria mellonella, or Bombyx mori. Among theseviruses, Autographa Californica (referred to in short as AcNPV) ispreferred.

In the production of a recombinant virus, a first recombinant vectorincorporating DNA regions which are not necessary for the multiplicationof the Baculovirus is first produced. In this case, it is necessary toplace, in this region, a promoter which functions in the Baculovirus,and it is preferable to insert a synthetic linker having an appropriaterestriction enzyme cleavage sequence downstream from this promoter.

Here, references to DNA regions which are not necessary formultiplication refer to regions which have no substantial effect on themultiplication of the virus even if they received mutations as a resultof the insertion of exogenous DNA, such as, for example, the polyhedringene of the Baculoviruses L. K. Miller, et al., Science, 219, pp.715-721 (1983)! and the like.

In this connection, it has been shown that polyhedrin is a protein witha molecular weight of approximately 29,000 daltons and that the genethereof is present in the Eco RI fragment of the AcVPV genome G. E.Smith, et al., J.Virol., 45, pp.215-225 (1983)!, and the DNA sequence ofthe polyhedrin gene was disclosed in the paper of G. E. Smith, et al.,Virology, 131, pp. 561-565 (1983)!.

Furthermore, with regard to promoters which function withinBaculoviruses, any base Sequence, whether synthesized or naturallyoccurring, which is capable of effectively functioning as a promoter ina transcription system possessed by a Baculovirus may be used; concreteexamples thereof include, for example, a gene promoter coding for apolyhedrin of a Baculovirus, a Baculovirus gene promoter coding for a10K polypeptide, and the like.

In order to increase the expression amount, it is preferable to use avector in which the polyhedrin gene promoter, the 5' non-translatedregion of the polyhedrin gene, a restriction enzyme cleavage sequencewhich is added immediately after the 5' non-translated region and the 3'non-translated region of the polyhedrin gene are connected in this orderand in which the polyhedrin structural gene sequence is completelyremoved. It has been shown that the expression amount of exogenous genesbecomes extremely large with this type of vector Matsuura, et al., J.Gen. Virol., 68, pp. 1233-1250 (1987); Japanese Unexamined PatentApplication, No. (Hei)1-285198!.

The cDNA of the HCV structural gene region used in the present inventionwas previously isolated by the present applicants (U.S. patentapplication Ser. No. 408,045), and furthermore, this type of HCV genefragment has been deposited at the Fermentation Research Institute bythe present applicants (pS7-28c: FERM BP-2638; pS1-713c: FERM BP-2637;and pU1-1216c: FERM BP-2594), and it is possible to use these genefragments as representative starting material.

The HCV-cDNA integrated into the expression vector may be an DNAfragment containing at least a section from the translation initiationcodon (ATG: methionine) of the precursor polypeptide coded for in theHCV genome to approximately the signalase recognition site (the 180th to190th amino acid, counting from methionine).

Furthermore, even in the case in which a longer section of cDNA, forexample, genes coding for up to approximately the 400th amino acid,counting from methionine, is expressed in cultured cells, it is possibleto conduct processing within host cells and to obtain the desired p22.

In this manner, at the time of the expression of p22 in cultured cells,in the case in which a large gene is used as the HCV structural gene tobe integrated, a polypeptide having a larger molecular size andpossessing the amino acids of the p22 peptide of the present inventionis partially obtained as a by-product. A specific reaction with bloodserum from human hepatitis C patients has also been confirmed, in thecase of an HCV antigen containing this type of p22 peptide and having alarger molecular weight. That is to say, the present invention providesan HCV antigen having a larger molecular weight and possessing the p22polypeptide as a portion thereof. With respect to the molecular weightof this polypeptide, the molecular weight varies in accordance with thelength of the HCV-cDNA which is integrated; for example, in the case inwhich HCV-cDNA having methionine as the N-terminus and coding for 289amino acids is used, a polypeptide of approximately 35 kilodaltons isobtained, and furthermore, in the case in which HCV-cDNA coding for 441amino acids is used, a polypeptide of approximately 50 kilodaltons isobtained.

(3) Detection of an HCV-related antibody using a recombinant HCVstructural protein containing p22

By means of the use of the recombinant HCV structural protein of thepresent invention as an antigen, it is possible to detect the HCVantibody which is specific thereto, and it is possible, by meansthereof, to make an early diagnosis of hepatitis C, which was notconventionally possible.

It is possible to use a common enzyme immunoassay (EIA), RIA,immunofluorescent technique, agglutination technique, or the like as themeasurement method.

That is to say, by means of the present invention, an antibody detectionmethod was established with respect to p22 derived from the HCVstructural gene, and diagnosis from blood sera and tissue samples hasthus become possible.

The p22 antibody cannot be detected in the blood sera of a healthyperson, a hepatitis A patient, or a hepatitis B patient, andfurthermore, the tendency for a positive result at a considerablyearlier stage of hepatitis C infection has been recognized, so that adetection method for the detection of this antibody can be used for theearly diagnosis of hepatitis C and for the screening of blood fortransfusions.

Hereinbelow, the present invention will be explained in detail withreference to examples; however, the present invention is in no waylimited to these examples.

EXAMPLES

(1) Construction of an Expression Plasmid in a COS Cell

The HCV structural gene was previously cloned by the present inventorsby means of the reverse transcription PCR method from Japanese non-A andnon-B hepatitis pathogen carriers, and this was made on the basis ofU.S. Pat. No. 5,372,928.

cDNA which was integrated into pS7-28c, pS1-713c, pS1-713g, andpU1-1216c plasmids was used in the expression of the HCV antigen proteinof the present invention. Using conventional methods, the base sequencesof the common portions of these 4 cDNA sections were cleaved withrestriction enzymes, and by means of recombining with other clones, aplasmid pS7/1-216 containing the cDNA of the entirety of these clonesconnected together was created. The accession numbers of each plasmidused and the various gene fragments which were used (cloned) in theplasmid pS7/1-216 which was constructed are shown.

    ______________________________________                                        PLASMID    ACCESSION NUMBER                                                                              FRAGMENT USED                                      ______________________________________                                        pS7-28c    FERM BP-2638    5' terminus -BsU 36I                               pS1-713g   None            Bsu 36I-Sal I                                      pS1-713c   FERM BP-2637    Sal I- Dde I                                       pU1-1216c  FERM BP-2594    Dde I-3' terminus                                  ______________________________________                                    

Furthermore, the HCV base sequence regions integrated into each plasmid,and the region integrated into the plasmid pS7/1-216 which wasconstructed, are shown below. The numbers used for the bases refer tothe case in which the initial base of the Japanese HCV cDNA of theplasmid pS7-28c is given the number "1".

    ______________________________________                                        PLASMID    HCV REGION   INTEGRATED REGION                                     ______________________________________                                        p57-28c    1-572        1-518                                                 pS1-713g   501-1100     519-874                                               pS1-713c   501-1100     875-1083                                              pU1-1216c  1063-1413    1084-1413                                             ______________________________________                                    

The pS1-713g plasmid has not been deposited; however, in comparison withthe pS1-713c plasmid, the G (guanine) at the 627th base is replaced byan A (adenine), and the T (thymine) at the 1041st base is replaced by aC (cytosine), and the amino acid sequence which is coded for isidentical in the case of both cDNA fragments.

The pS7/1-216 sequence prepared in this manner contains the 1413 basesof the HCV cDNA from Japanese patients, and at the 3' end thereof, a BamHI linker and an Eco RI linker are attached. In this cDNA, an openreading frame exists which occupies almost the entire length thereof,and at the 91st base thereof, an ATG sequence, which is thought to be atranslation initiation codon, was located.

Next, this pS7/1-216 sequence was cleaved at the Acc I site located 12bases upstream from the translation initiation codon, and then a Pst Ilinker was attached thereto. Next, a further Kpn I linker was added atthe Eco RI site which was attached to the 3' end of the cDNA, the cDNAwas recovered as a Pst I-Kpn I fragment, and this was cloned at the PstI-Kpn. I site of the expression vector pcDL-SRα 296 Yutaka Takebe, etal., Mol. Cell Biol., 8, 466-472, (1988)! in a cultured cell system.This expression vector was developed by Takebe and colleagues, andcomprises a strong promoter, splicing sequence, and polyA sequence. Theexpression plasmid pSR316 was prepared in this manner (see FIG. 1).

Using pSR316 as a base, 2 types of plasmids which lacked a portion ofthe HCV structural gene, pSR312 and pSR316d46, were prepared (see FIG.1).

A section was removed to prepare the pSR312 plasmid by means of cuttingwith Eco RI downstream from the Pvu I site at the 957th base of the HCVcDNA sequence and the subsequent synthesis of a new strand and bondingwith polymerase.

In the pSR316d46 plasmid, the 201 bases from 210-411 are deleted bymeans of the partial cutting of the HCV cDNA sequence at the Apa I sitesat the 210th and 411th bases and ligating with a ligase. In this case,the translation frame of the HCV cDNA is maintained, and it is believedthat a deletion of 67 amino acids is caused.

(2) Expression of p22 in COS Cells

20 micrograms of each of the above 3 plasmids were transfected by meansof a calcium phosphate method into 2×10⁶ COS-1 cells of a cultured cellline from simian kidney cells Y. Gluzuman, Cell, 23,pp. 175-182 (1981)!.After 2 days, using the blood serum of chronic hepatitis C patients, theantigens were stained by means of an immunofluorescence techniquefollowing normal methods. Furthermore, on the same day, intracellularproteins were detected by means of Western blot method and using theabove-described blood serum of chronic hepatitis C patients.

During the immunofluorescence technique, in approximately 10% of thecells, antigens which were localized in the cytoplasm and which werestained in a fine granular form were observed. This fluorescence wasdetected in equal degrees when the pSR316 and pSR312 plasmids were used;however, in the case of the pSR316d46 plasmid, almost no fluorescencewas detected, and furthermore, only in the case of the vector pcDL-SRα296, into which no HCV-cDNA was integrated, was no fluorescencedetected.

When the pSR316 plasmid was used in the Western blot method, a proteinhaving a molecular weight of 22 kilodaltons was detected as an extremelythick band (see FIG. 2). This band was also detected in the case of thepSR312 plasmid; however, it was not detected in the case of thepSR316d46 plasmid or in the case of the vector pcDL-SR α 296, so that itis believed that this represents detection of the antigen which wasobserved using the immunofluorescence technique. Irrespective of thefact that the length of the translatable region from the JapaneseHCV-cDNA which was used was 441 amino acids in the case of the pSR316plasmid, and 289 amino acids in the case of the pSR312 plasmid, the factthat the molecular weight of the main protein which was detected was 22kilodaltons is believed to be caused by the processing of the proteinswithin the COS cells after the translation of these proteins. The sametypes of mechanisms have been observed in the proteins of the structuralgene regions of the Flaviviruses, which are believed to be related tothis virus C. M. Rice, et al., Science, 229, pp. 726-733 (1985)!, and itis believed that the precursor peptide translated from this gene wascleaved with the enzyme signalase within the cells.

From the fact that p22 was detected in the case of the pSR316 and pSR312plasmids, but not in the case of the pSR316d46 plasmid, it is believedthat the region coding for p22 lies between the initial Met (methionine)of the amino acid sequence, and the 289th amino acid therefrom, and itis further believed that the region lacking in the pSR316d46 plasmid isincluded in this code region. Furthermore, a signal peptide typesequence is observed which comprises a series of hydrophobic amino acidsfrom the 180th to the region of the 190th in the amino acid sequencewhich is coded for, and it is conjectured that this is the signalasecleaving recognition site. Furthermore, in the pSR316d46 plasmid, a 14kilodalton protein was detected as a weak band; however, it is believedthat this represents the detection of a protein which is shortened by 67amino acids as a result of a deletion of 201 bases from the originalp22.

Furthermore, in the Western blots, with respect to minor proteins otherthan p22, a band was detected at approximately 50 kilodaltons when thepSR316 plasmid was used, and a band was detected at approximately 35kilodaltons when the pSR312 plasmid was used. From the fact that thesebands correspond nearly exactly to the sizes of the proteins of thecomplete code regions of the HCV peptides contained in the plasmids, itis believed that these bands represent, in all likelihood, precursorproteins translated from the full length of the cDNA used in theexpression.

(3) Expression Using a Baculovirus

The ends of a cDNA fragment (AccI site cleaved-EcoRI) which is identicalto that used in the construction of the pSR316 plasmid were made bluntby means of polymerase processing, and a pAC316 plasmid was constructedby means of cloning the cDNA fragment at the Bam HI site of a pACYM1plasmid Matsuura, et al., J. Gen. Virol., 68, pp. 1233-1250 (1987)!having ends which were made blunt by means of the same polymerase.

After this, this plasmid was co-transfected together with BaculovirusDNA into cultured cells from an insect Spodoptera frugiperda cells!, anda recombinant Baculovirus was isolated following normal methods.

Sf cells were transfected with this virus in MOI10, and the expressionof p22 was determined after 72 hours had elapsed. 1×10⁴ transfectedcells were migrated using SDS-PAGE (SDS-polyacrylamide gelelectrophoresis), and after this, when staining was conducted withCoomassie Brilliant Blue (CBB), a 22 kilodalton protein was detected asan expression protein. This band was not detected in the originalBaculovirus-transfected cells into which the HCV cDNA was notintegrated. Furthermore, in the immunofluorescence technique in whichthe blood serum of chronic hepatitis C patients was used, fluorescencewhich was specific to the cytoplasm was detected in 100% of thetransfected cells. In the detection by means of the Western blot method,a 22 kilodalton protein was detected in this expression system as a mainexpression product. From the fact that the apparent molecular weight ofthis protein does not vary as a result of the addition of tunicamycin,which is identical to the case of COS cells, it is believed that theN-sugar chain is not attached. Furthermore, it is clear that theisoelectric point is extremely basic, so that the 22 kilodalton proteinwhich was detected here cannot be distinguished within the parameters ofthe detection method from the p22 which was identified in the COS cells.

(4) HCV Antibody Detection Method Using a Recombinant HCV StructuralProtein

HCV structural protein expressing cells (AeHCV-SF9) which were infectedwith the above recombinant Baculovirus were recovered, were rinsed twicewith PBS, and then were suspended in 50 mM of Tris-hydrochloride buffer(pH 8.0) having added thereto 2 mM of EDTA and 0.1 mM of DTT so as tocreate a density of of 5×10⁶ cells/ml.

This cellular suspension fluid was lysed by sonication, and by means ofcentrifugal processing (12000×G, 20 minutes, 4 degrees Celsius) theclear supernatant thereof was recovered. To this was added saturatedammonium sulfate so as to reach 33% saturation, centrifugation was againconducted, 1 ml of PBS was added to the precipitate fraction thereof per5×10⁶ cells, this was dissolved by sonication, and HCV antigen proteinwas obtained by means of decanting. This was maintained in a frozenstate at a temperature of -80 degrees Celsius, and in the case in whichthis was to be used as an antigen for an immunoassay, it was coated ontoan ELISA plate immediately after ultrasonic processing.

The above crudely produced HCV antigen was diluted 50 times with PBS,and 100 microliters of this was added to each of the wells of a 96 wellmicrotiter plate, this was stored overnight at a temperature of 4degrees Celsius, and the ELISA plate was thereby coated.

This plate was twice washed with PBS-Tween, 200 microliters of PBScontaining 3% skim milk was added to each well, this was incubated for 1hour at room temperature, and blocking was thereby conducted.

100 microliters of the blood serum which was the object of the HCVantibody assay was placed in each well of the plate, and this was storedfor 2 hours at room temperature.

This plate was washed 4 times with PBS-Tween, and to each well was added100 microtiters of alkaline phosphatase-labeled goat anti-humanimmunoglobulin diluted with PBS-Tween with 3% skim milk added thereto,and this was incubated for 1 hour at room temperature.

This plate was then washed 4 times with PBS-Tween, and to each well wasadded 700 microliters of alkaline phosphatase substrate solution.

This was left to stand for 1-2 hours at room temperature, and when theA405 absorption of the positive reaction reached a value ofapproximately 1.5, the absorbance of all the wells was measured.

Using this assay, the blood sera of actual patients were tested, and thefollowing results were obtained.

In 2 typical cases of post-transfusion a non-A, non-B hepatitis, bloodwas collected at intervals starting immediately after the onset ofsymptoms, and hepatic function tests, the test for the presence ofanti-C100 antibodies, which is presently the only test agent for HCVantibodies, and tests for the presence of an antibody to the p22 peptidewere conducted. During a 12 month period of observation, an increase inthe p22 antibodies which was essentially simultaneous with an increasein the GPT value was noted, even in the case in which anti-C100antibodies were not detected.

Furthermore, in the other cases, the anti-C100 antibodies increased 4months after the transfusion; however, antibodies to p22 were detected 3months prior to this, that is to say, nearly simultaneously with theonset of symptoms. A result identical to the detection of the antibodywas achieved by means of Western blot method using the above protein.

Next, the blood sera of 5 healthy people and the blood sera of non-A,non-B hepatitis patients (of which 6 tested positive for the anti-C100antibody and 4 tested negative for the anti-C100 antibody) were testedtogether. Of the healthy persons, none tested positive, while of thenon-A, non-B hepatitis patients who tested positive for the anti-C100antibody, 5 out of 6 tested positive, and of the non-A. non-B hepatitispatients who tested negative for the anti-C100 antibody, 4 out of 4tested positive.

From the above results, the following has been confirmed.

1 This antibody cannot be detected in normal subjects.

2 90% of non-A, non-B hepatitis cases test positive.

3 Even in cases of sera of the patients who were clinically diagnosed asnon-A, non-B hepatitis which tested negatively for the anti-C100antibody, approximately 100% test positive according to this method.

4 Even with blood serum which tests positive for the anti-C100 antibody,there are cases in which the test for this antibody is negative.

From these results, it has been confirmed that the antibody to p22 isspecifically detectable in cases of present or past HCV infection. Thisantibody is induced at an early stage of infection, so that thedetection thereof is extremely useful for an early diagnosis ofhepatitis C.

What is claimed is:
 1. A recombinant hepatitis C virus antigenpolypeptide obtained by:expressing pSR312 or pSR316 plasmid in COScells, and isolating an expressed protein which has a molecular weightof approximately 22 kilodaltons, as determined by SDS-PAGE.
 2. Arecombinant hepatitis C virus (HCV) antigen polypeptide which has amolecular weight of approximately 22 kilodaltons as determined bySDS-PAGE, said antigen polypeptide is expressed from the hepatitis Cvirus structural gene region in animal cells transfected with HCV DNA,said HCV DNA comprises from the translation initiation codon of saidstructural gene region to at least the 180th amino acid encoded by saidstructural gene region, and said antigen polypeptide is specificallyrecognized by antibodies present in sera of HCV infected patients.